The present invention relates to the field of ribosomal antimicrobial substances. The microbial ribosome is the target for many important antimicrobial substances, e.g. antibiotics. These compounds interfere with essential steps of protein synthesis. Ribosomal antimicrobial substances interfere with the microrganism's protein synthesis in such a manner that its propagation is reduced or it is killed. Today, the term antimicrobial substance is understood to encompass all substances that are active against microorganisms of any origin such as bacteria, protozoa and fungi. Ribosomal antimicrobial substances, in particular ribosomal antibiotics comprise, for example, 2-deoxystreptamine aminoglycosides, compounds of the streptomycin family, macrolides, lincosamides and streptogramin B.
Microbial resistance is a common problem with antimicrobial therapy these days. Recent reports indicate that resistance may be the result of one or more mutation(s) within the ribosomal RNA. The investigation of these mutations has proven difficult because microorganisms, e.g. bacteria, generally have several gene copies of rDNA per genome. However, new mutational strategies employing bacteria with low rRNA operon copy numbers such as Mycobacterium smegmatis allow for the study of the molecular basis of antimicrobial substance resistance. (Sander et al. Introducing mutations into a chromosomal rRNA gene using a genetically modified eubacterial host with a single rRNA operon, Molecular Microbiology (1996) 22(5), 841-848).
For example, the following positions were identified to confer resistance to antimicrobial substances, e.g. antibiotics: 1408 (Sander, supra), and 1491 (Pfister at al. Mutagenesis of 16 S rRNA C1409-G1491 base-pair differentiates between 6′OH and 6′NH3+ aminoglycosides, J. Mol. Biol. (2005) 346: 467-475)) in 16sRNA and position 2058 in 23 S rRNA (Sander et al. Mol. Microbiol. 1997, 26: 469-484). Specifically, the mutations A1408G, G1491C(A) and A2058G confer resistance depending on the antimicrobial substance employed. All reference numbers indicating nucleotide positions are based on the numbering of E. coli. 
The corresponding positions in eukaryotic cytosolic rRNA are 1408G, 1491A, and 2058G, while the same position in eukaryotic mitochondrial rRNA are 1408A, 1491C, and 2058G.
From the above it becomes evident, that cytosolic and mitochondrial ribosomes may be susceptible or resistant to antimicrobial activity depending on the drug and the rRNA composition.
It has been suggested (Böttger et al., Structural basis for selectivity and toxicity of ribosomal antibiotics, EMBO reports, (2001) Vol. 2:4, 318-323) that                “The in vitro and in vivo selection of drug resistant bacteria, mapping of resistance conferring mutations and comparison to eukaryotic (mitochondrial and cytoplasmic) ribosomal nucleic acid and protein sequences may offer an important strategy to predict the specificity and toxicity of future antibiotics targeting the bacterial ribosome.”        
At present, antimicrobial substances, in particular antibiotics are tested for antimicrobial activity and resistance by contacting microorganisms directly whereas toxicity is tested by contacting eukaryotic cells and animals.
It is the object of the present invention to provide a faster, more economical, and simpler method for determining the specificity and toxicity of candidate antimicrobial substances. This object is solved by providing a method for identifying ribosomal antimicrobial substances being selective for microbial but not for mitochondrial and/or cytosolic ribosomes, comprising the following steps:                a) providing                    (i) at least one bacterial strain with microbial ribosomes, and            (ii) at least one bacterial strain with chimeric mitochondrial bacterial ribosomes, and/or            (iii) at least one bacterial strain with chimeric cytosolic bacterial ribosomes;                        b) contacting a candidate ribosomal antimicrobial substance with each of the bacterial strains according to a);        c) determining an interaction of the candidate ribosomal antimicrobial substance with one or more of the ribosomes of each of the bacterial strains according to a).        
It was found that microbial systems comprising (i) microbial, preferably bacterial, (ii) chimeric mitochondrial bacterial and/or (iii) chimeric cytosolic bacterial ribosomes are sufficient for determining the ribosomal specificity and, consequently, the toxicity of ribosomal antimicrobial substances. The method of the present invention relies completely on bacterial cells and avoids the eukaryotic cell systems and animal models that were employed until now for specificity and toxicity testing. Bacterial systems are much more economical from a point of costs, replication time, manipulation and handling than their eukaryotic counterparts. It was demonstrated that the results obtained with purely bacterial strains significantly coincide with the results obtained with eukaryotic cell systems and animals.
In a preferred embodiment of the method of the present invention at least one of the bacterial strains mentioned in sections (i) to (iii) above may be substituted by a functionally equivalent cell-free biological system. More preferably, at least two, most preferably all three bacterial strains mentioned in sections (i) to (iii) above are substituted by a functionally equivalent cell-free biological system. In this respect it is to be noted that the term “bacterial strain” is meant to encompass functionally equivalent cell-free biological systems. The term “cell-free system” is well understood by those skilled in the art and does not require any further comment. Also, methods for providing cell-free systems are common general knowledge in the art and are routinely practiced by those of average skill in the art. The term “functionally equivalent” in the above respect is meant to relate to cell-free systems that comprise at least a functional ribosomal system capable of translational activity for producing polypeptides from nucleotides.
The term “ribosomal antimicrobial substance” is meant to define compounds that specifically interact with microbial, preferably bacterial ribosomes and function as antimicrobial, preferably bacteriocidal and/or bacteriostatic agents.
Ribosomal antimicrobial substances selective for microbial, preferably bacterial ribosomes, according to the present invention are those that do not affect mitochondrial and/or cytosolic ribosomes, preferably those that do not affect mitochondrial and cytosolic ribosomes.
The term “microbial ribosome” includes wild-type and chimeric ribosomes of micro-organisms, such as fungal, bacterial and protozoic ribosomes.
Antibiotics have been shown to be effective ribosomal antimicrobial substances in various microbial organisms such as, e.g. the helminth Echinococcus multicularis (Mathis et al. Antimicrobial Agents and Chemotherapy, August 2005: 3251-3255) and the protozoan Acanthamoeba castellanii (Mathis et al. Molecular & Biochemical Parasitology, 135, 223-227, 2004). Hence, the method of the invention is suitable for identifying ribosomal antimicrobial substances, in particular antibiotics, selective for many, most or even all types of microbial organisms. For example, the method of the present invention is suitable for identifying antimicrobial substances that are selective for the parasites giving rise to leishmaniasis and trypanosomiasis.
The term “chimeric mitochondrial bacterial ribosome” and the term “chimeric cytosolic bacterial ribosome” are meant to define a ribosome which is derived from a bacterial ribosome, wherein one or more nucleotide(s) of the ribosomal RNA has/have been replaced by the corresponding nucleotide from a mitochondrial ribosome or a cytosolic ribosome, respectively.
Typically, the contacting in step b) of the method of the present invention is effected by adding at least one candidate antimicrobial substance, optionally in a solvent or solvent system, to the bacteria or the functionally equivalent cell-free biological system directly, e.g. placing it onto the surface of the bacteria or cell-free system, or indirectly, e.g. adding the antimicrobial substance to the liquid or solid medium (e.g. gel, beads) surrounding or underlying the bacterial strain(s) or cell-free system(s). The contacting step is non-limiting and may be effected in any suitable manner known to the person skilled in the art.
The selectivity of the candidate antimicrobial substance and, consequently, the toxicity with regard to mitochondrial or cytosolic ribosomes may be determined by routine methods, in particular by comparing the candidate antimicrobial substance' inhibitory activity on the growth of bacterial strains and/or functionally equivalent cell-free ribosomal biological systems comprising (i) microbial ribosomes, and (ii) chimeric mitochondrial bacterial ribosomes, and/or (iii) chimeric cytosolic bacterial ribosomes.
In a preferred embodiment the method of the invention is one for identifying ribosomal antimicrobial substances selective for bacterial, protozoic and/or fungal ribosomes, preferably selective for bacterial and/or protozoic ribosomes, more preferably selective for bacterial ribosomes. In a particularly preferred embodiment, the method of the present invention is suitable for identifying antimicrobial substances selective for the protozoa giving rise to leishmaniasis and/or trypanosomiasis.
In a more preferred embodiment of the method of the invention the at least one bacterial strain (or functionally equivalent cell-free system) with microbial ribosomes of step a) (i) comprises microbial ribosomes selected from the group consisting of natural or chimeric bacterial, protozoic and fungal ribosomes, preferably natural or chimeric bacterial and/or protozoic ribosomes, more preferably natural or chimeric bacterial ribosomes. The bacterial strain (or functionally equivalent cell-free system) comprises at least one of said microbial ribosomes and may comprise microbial ribosomes of more than one microorganism, e.g. fungal and bacterial, fungal and protozoic, protozoic and bacterial or protozoic, fungal and bacterial ribosomes.
Preferred candidate ribosomal antimicrobial substances are selected from the group consisting of aminoglycosides, macrolides, lincosamides, preferably aminoglycosides, more preferably 2-deoxystreptamines.
Although the microbial ribosomes may be any bacterial ribosomes when antimicrobial substances selective against bacteria, i.e. antibiotics, are desired, it is preferred that the microbial ribosomes for use in the method of the invention are bacterial ribosomes from Mycobacterium smegmatis. 
For identifying ribosomal antimicrobial substances selective against fungi it is preferred that the microbial ribosomes are chimeric fungal ribosomes characterized in that they are derived from bacterial ribosomes comprising at least one mutation selected from the group consisting of 1408A, 1410G, 1490C, and 1491G of according to the numbering of E. coli. 
For identifying ribosomal antimicrobial substances selective against protozoa it is preferred that the microbial ribosomes are chimeric protozoic ribosomes characterized in that they are derived from bacterial ribosomes comprising at least one mutation selected from the group consisting of 1408A, 1409U, 1409A, 1410G, 1410 A, 1490U, 1491G, 1491U and 2058A of according to the numbering of E. coli. 
Preferred chimeric mitochondrial bacterial ribosomes for practicing the method of the present invention are characterized in that they are derived from bacterial ribosomes comprising at least the mutation of G1491C according to the numbering of E. coli, preferably having further mutations selected from the group consisting of G1410C, U1411C, C1412U, A1413C, U1414C, G1415U, A1416C, U1484G, U1485A, G1488A, A1489G and C1490A.
More preferably, the chimeric mitochondrial bacterial ribosomes comprise mutations G1410C, U1411C, C1412U, A1413C, U1414C, G1415U, A1416C, U1484G, U1485A, G1488A, A1489G and C1490A.
Most preferably, the chimeric mitochondrial bacterial ribosomes are further humanized by mutation(s), preferably by mutation(s) in the ribosomal decoding site.
Preferred chimeric cytosolic bacterial ribosomes for practicing the method of the present invention are characterized in that they are derived from bacterial ribosomes comprising at least the mutation(s) of A1408G and/or G1491A according to the numbering of E. coli, more preferably having further mutations selected from the group consisting of G1410U, U1411A, A1413U, U1414A, G1415C, A1416C, U1484G, U1485G, G1486A, G1487A, A1489U, and C1490A.
More preferably, the chimeric cytosolic bacterial ribosomes comprise mutations G1410U, U1411A, A1413U, U1414A, G1415C, A1416C, U1484G, U1485G, G1486A, G1487A, A1489U, and C1490A, and much more preferably further comprise A1408G and G1491A.
Most preferably, the chimeric cytosolic bacterial ribosomes are further humanized by mutation(s), preferably in the ribosomal decoding site.
It is noted that the method of the invention is not limited to microbial ribosomes featuring mutations in the 16S rRNA much less is it limited to mutations in the decoding site of 16S rRNA. The embodiments herein relating to mutations in the decoding site of 16S rRNA merely serve as examples for illustrating the present invention and are by no means meant to limit its scope. For example, the method of the present invention encompasses any manipulations of functionally relevant regions of 16S rRNA or 23S rRNA that have a natural sequence polymorphism that can serve as a mutational basis for the method of the invention.
While there are a number of standard methods for determining the interaction of ribosomal antimicrobial substances available to those skilled in the art, it is preferred that for practicing the method of the invention the interaction of the ribosomal antimicrobial substance is determined by calculating the minimal inhibitory concentration (MIC) of the antimicrobial substance in each of the bacterial strains and/or cell-free systems according to step a), preferably the MIC of the antimicrobial substance relative to the MIC of the bacterial strain and/or cell-free system (i).
In a preferred embodiment the candidate antimicrobial substance identified according to the present invention is selective for bacterial, fungal and/or protozoic but not for mitochondrial and cytosolic ribosomes. More preferred it is selective for bacterial ribosomes, i.e. a ribosomal antibiotic.
In order to avoid interference of the bacterial cell's (or cell-free system's) own naturally occurring ribosomes with the introduced at least partially heterologous, mitochondrial or cytosolic bacterial ribosomes, at least part of at least one gene encoding the naturally occurring ribosomes can be modified, replaced or deleted.
In a preferred embodiment the method according to the invention is one, wherein the                (i) at least one bacterial strain with microbial ribosomes, and/or        (ii) at least one bacterial strain with chimeric mitochondrial bacterial ribosomes, and/or        (iii) at least one bacterial strain with chimeric cytosolic bacterial ribosomes;is a bacterial strain, wherein at least part of at least one gene encoding the naturally occurring bacterial ribosomal RNA sequence has been deleted and replaced by at least part of at least one gene encoding a heterologous microbial (i), mitochondrial (ii) and/or cytosolic (iii) ribosomal RNA sequence, respectively. Again, instead of said bacterial strains equivalent cell-free biological systems can be employed.        
More preferably, the bacterial strain for practicing the method is Mycobacterium smegmatis, wherein at least part of the rrnA and/or rrnB gene(s) has (have) been deleted and replaced by at least part of at least one gene encoding a heterologous microbial (i), mitochondrial (ii) and/or cytosolic (iii) ribosomal RNA sequence.
In another preferred embodiment the cell-free system for practicing the invention is a system comprising bacterial ribosomal components, preferably from Mycobacterium smegmatis, preferably comprising its ribosome(s), wherein at least part of the chromosomal rRNA, preferably rrnA and/or rrnB gene(s), has (have) been deleted and replaced by at least part of at least one gene encoding a heterologous microbial (i), mitochondrial (ii) and/or cytosolic (iii) ribosomal RNA sequence.
Another aspect of the present invention relates to the use of bacterial strains or functionally equivalent cell-free biological systems in a method according to the invention.
In a preferred embodiment of this aspect, the invention relates to the use of bacterial strains or functionally equivalent cell-free biological systems comprising chimeric fungal ribosomes characterized in that they are derived from bacterial ribosomes, wherein the ribosomes are characterized in that they are derived from bacterial ribosomes comprising at least one mutation selected from the group consisting of 1408A, 1410G, 1490C, and 1491G of according to the numbering of E. coli in a method according to the invention.
In another preferred embodiment of this aspect, the invention relates to the use of bacterial strains or functionally equivalent cell-free biological systems comprising chimeric protozoic ribosomes characterized in that they are derived from bacterial ribosomes, wherein the ribosomes are characterized in that they are derived from bacterial ribosomes comprising at least one mutation selected from the group consisting of 1408A, 1409U, 1409A, 1410G, 1410 A, 1490U, 1491G, 1491U and 2058A of according to the numbering of E. coli in a method according to the invention.
In another preferred embodiment of this aspect, the invention relates to the use of bacterial strains or functionally equivalent cell-free biological systems comprising chimeric mitochondrial bacterial ribosomes characterized in that they are derived from bacterial ribosomes comprising at least the mutation of G1491C according to the numbering of E. coli, preferably having further mutations selected from the group consisting of G1410C, U1411C, C1412U, A1413C, U1414C, G1415U, A1416C, U1484G, U1485A, G1488A, A1489G and C1490A in a method according to the invention.
More preferably, the chimeric mitochondrial bacterial ribosomes comprise mutations G1410C, U1411C, C1412U, A1413C, U1414C, G1415U, A1416C, U1484G, U1485A, G1488A, A1489G and C1490A.
Most preferably, the chimeric mitochondrial bacterial ribosomes are further humanized by mutation(s), preferably in the ribosomal decoding site.
In a further preferred embodiment of this aspect, the invention relates to the use of bacterial strains or functionally equivalent cell-free biological systems comprising chimeric cytosolic bacterial ribosomes characterized in that they are derived from bacterial ribosomes comprising at least the mutation of A1408G and/or G1491A according to the numbering of E. coli, more preferably having further mutations selected from the group consisting of G1410U, U1411A, A1413U, U1414A, G1415C, A1416C, U1484G, U1485G, G1486A, G1487A, A1489U, and C1490A in a method according to the invention.
More preferably, the chimeric cytosolic bacterial ribosomes comprise mutations G1410U, U1411A, A1413U, U1414A, G1415C, A1416C, U1484G, U1485G, G1486A, G1487A, A1489U, and C1490A, and more preferably further comprise mutations A1408G and G1491A.
Most preferably, the chimeric cytosolic bacterial ribosomes are further humanized by mutation(s), preferably in the ribosomal decoding site.
As mentioned before, in order to avoid interference of the bacterial cell's (or cell-free biological system's) own naturally occurring ribosomes with the introduced at least partially heterologous, mitochondrial or cytosolic bacterial ribosomes, at least part of at least one gene encoding the naturally occurring ribosomes can be modified, replaced or deleted.
Therefore, in another preferred embodiment the present invention relates to the use of a bacterial strain or functionally equivalent cell-free biological system, wherein at least part of at least one gene encoding the naturally occurring bacterial ribosomal RNA sequence has been deleted and replaced by at least part of at least one gene encoding a heterologous microbial (i), mitochondrial (ii) and/or cytosolic (iii) ribosomal RNA sequence in a method according to the invention.
More preferably, the present invention is directed to the use according to the invention, wherein the bacterial strain is Mycobacterium smegmatis or wherein the functionally equivalent cell-free biological system is derived from Mycobacterium smegmatis, wherein at least part of the rrnA and/or rrnB gene(s) has (have) been deleted and replaced by at least part of at least one gene encoding a heterologous microbial (i), mitochondrial (ii) and/or cytosolic (iii) ribosomal RNA sequence.
A further aspect of the present invention relates to a kit of parts comprising any one or more of the above bacterial strains or functionally equivalent cell-free biological systems or components thereof for use in the method of the present invention and optionally written instructions, buffer materials, bacterial nutrient(s), aqueous solvent(s), antimicrobial substance(s), etc.
In the following specific embodiments of the present invention will be described for the purpose of illustrating the present invention and for providing a best mode for carrying out the invention.